The incorporation of unnatural chemical groups into proteins has increasing importance in protein science and cell biology, and the biosynthesis of proteins containing unnatural amino acids can expand the structural and chemical diversity in proteins. One method of incorporating unnatural amino acids into proteins includes microinjecting chemically acylated tRNA and UAG-containing mutant mRNA into cells. Unfortunately, because this method involves microinjection, the technique is limited mainly to large Xenopus oocytes, and it is not suitable for studies that require large numbers of cells. Moreover, the tRNA is chemically acylated with the unnatural amino acid in vitro, and the acylated tRNA is consumed as a stoichiometric reagent during translation and cannot be regenerated. Therefore, yields of mutant proteins are low and long periods of data collection are not feasible.
Genetically encoding unnatural amino acids in cells can be used to study proteins in their native environment within the cell. One such method for expanding the genetic code to include unnatural amino acids was developed in E. coli (Wang et al., (2001) Science 292, 498-500). This method involved the generation of a new tRNA/aminoacyl-tRNA synthetase pair that was specific for an unnatural amino acid, and that decoded a blank codon unused by a common amino acid (such as a stop codon or extended codon). The tRNA/synthetase pair worked with the protein biosynthesis machinery of the host cell, and did not crosstalk with endogenous multiple tRNA/synthetase pairs.
However, genetically encoding unnatural amino acids in eukaryotes is more complicated because eukaryotic cells (including mammalian cells) and E. coli differ significantly in tRNA transcription, processing and transportation, leading to inefficient biosynthesis of orthogonal prokaryotic tRNAs in mammalian cells. If it were possible to genetically encode unnatural amino acids in eukaryotic cells, for instance yeast or mammalian cells, such a method would be a powerful tool in fields such as protein science, neuroscience, and cell biology.